Trienoic fatty acids and plant tolerance of temperature

The biophysical reactions of light harvesting and electron transport during photosynthesis take place in a uniquely constructed bilayer, the thylakoid. In all photosynthetic eukaryotes, the complement of atypical glycerolipid molecules that form the foundation of this membrane are characterised by sugar head-groups and a very high level of unsaturation in the fatty acids that occupy the central portion of the thylakoid bilayer. alpha-linolenic (18:3) or a combination of 18:3 and hexadecatrienoic (16:3) acids typically account for approximately two-thirds of all thylakoid membrane fatty acids and over 90% of the fatty acids of monogalactosyl diacylglycerol, the major thylakoid lipid [1, 2]. The occurrence of trienoic fatty acids as a major component of the thylakoid membrane is especially remarkable since these fatty acids form highly reactive targets for active oxygen species and free radicals, which are often the by-products of oxygenic photosynthesis. Photosynthesis is one of the most temperature-sensitive functions of plant [3, 4]. There remains a widespread belief that these trienoic fatty acids might have some crucial role in plants to be of such universal occurrence, especially in photosynthesis tolerance of temperature [5].

plants in which the gene encoding the chlora-pIast (1)-3 fatty acid desalurase (FAD7) WolS silen· ceci. Th~tobatco transformant contaÎnecl reduced level of trienoic falty acids. Mulant photosynthesis activity WolS higher alter brief high temperature treatments and mutant plants adapte<! better to high temperature compared to the non·translormed wlld type tobacco. Thtst observations led to Ihe proposai that plants may adapt 10 temperature changes by a1tering the fatty acid composition of their membrane lipids.
Risumi: les membranes des chloroplastes, ' >iege tk la phorosynlMse, sont lm ricM en ocitm gras tri·inwfuris. la proportion de Cts odtft"> gras dons ks mtmbro~végétales~f~ff! modikxs oechangements de lemp€rotUft. Poorcette misan. il aété postuli que ces acides gras sont impfiquis dons ln mkonismn de rêslstance il la cholevr OU ou froid de5 fonctions membfO'noires. en patticulitr de la pholosynlhtse qui est Ufl(' dt1 f<JncOOns rk 10 ceilu1e les plus strnibIts il 10 ftm· The biophysîcal (faCtionS of light harwsting and electron transport during photosynthesîs take place in a uniquely constructecl bilayer, the thylakoîd. In ail photosynthetic eukaryotes, the complement of atypical glycerolipid molecules that form the foundalion olthis membrane are characterised by sugar head-groups and a very high level of unsaturation in the fatty acids that occupy the central portion of the thylakoid bilayer. lX-linolenic {18:3} or a combination of 18:3 and hexilde<atrienoic (16:3) acids typically account for approximately two-thirds of ail thy. Iakoid membrane fatty acids and over 90% of the fatty acids of monogalactosyl diacylglyce-roI, Ihe major thylakoid lipid [1,2]. The occurrence of trienQic fatty acids as a major component of the thylakoid membrane is especially remarkable since these fatty acids fOfm highly reactivt targets for active oxygen species and free radica!s, which are often the by-products of oxygenic photosynthesis. Pholosynthesis is one of the mest t~ture-sensîtîve functions of plant [3,4]. There remains a widespread beIief that these trienoic fatty acids might have sorne crucial role in plants to be 01 such universal occurrence, especially in photosynthesîs tolerance ollemperature {5J.
Do plants adapt to temperature by changlng the faUy acld composition of thelr membrane IIplds? ln sorne deserl and evergr~n pianu, the pro· portion of tri€noic fatty acids in the membrane glyceralipids decreases when planls acclimate ta high temperalures [6]. Canversely, cald accli· mation in higher plants induces increases in trienajc faity acid level in leaves [7). These fatty acid content changes have also been shown to be associale<! with enhanced chilling or heal tolerance in mutants 01 Arobidopsis or cyano· bacteria [review in: [8][9][10][11] and in transformed toba<:co ptants [12,13) ways in plant cel1s for the biosynthesis of gJyce-roIipids and the associated production of potyunsaturated fatty acids [17]. 80th pathways are initîattd by the synthesis of a 16:o-acyl carrier proteÎn (ACP) in the plastid by the fatty acid syn-\hase. This 16:O-ACP may be eIongated to 18:0-ACP and Ihen desaturaltd 10 18: l-ACP by a soluble desaturase so that 16:0-ACP and 18:1-ACP are the primary products of plastid latty acid synthesis. There produCls are eilher used directly in the prokaryotic pathW4Y localed in the chloroplast inner envelope for the synthesis of the glycerolipid components 01 the chloro· piast membranes or exportecl from the chloropiast as CoA thÎoeslers and incorporated into phosphalidykholine and other lipids in the endoplasmic reli<ulum by the eukaryotic pathway. In addition, the diacylglycerol moiety of phospha. tidylcholine can be retumed to the chloroplast enl'elope and used as a second source 01 precurson for the synthesis of chloroplast gly<erolipids. ln each pathway, further desaturation of 16:0 and 18:1 occurs only after these fatty acids have been incorporated into the major mem· brane lipids. In Arobidops~, three gene products, FAD3, FAD7, and fADS, mediate the synthesis of trienoK: latty acids from 18:2 and 16:2. The FAD3 gene produet~an endoplasmic reticulum desalurast. The FA07 and fAD8 gene5 encode two d*:ropIasl isozymes that recognise as a substrate either 18:2 (J( 16:2 attached to any of the chloroplast lipids. Amutation in one of these three genes results in no more than a partial reduction of the trienoic fatly acid content. On its OW'O, the ,adj mutation reduces the desatuoa. 1IOL-9 tr I.lAtMERIF!VRlfRlOO2 43 ration level of the thytakoid galactolipids only marginally. The fadJ mutation results in a lemperature-<lependent reduction in the 18:3 and 16:3 content in thyIakoid-specifIC Ieaflipids 118L whereas the fatty acid composition of fad8 is indistinguishable from wild type [19). To obtain a more pronounced altetation in the trienoic fatty acid contenl. it has been necessary to generate multiple mutant lil'leS [20]. Atriple mutant line of ArobOOpsis. fDdJ·}1odJ·} fada, hM been produced that is completely deflcient in \8:3 and 16:3 fatty atids either in the thyIakoid Of any other membrane of the cell ( Figure J).  suring the fluorescence parameter FjFm' or in the light by measuring lfl. under physiologieal lighllevels (PFD of 100~mol quanta m-l S-I). Figure 3 shows that both wild type and fodJ·2 fad7·} fad8 mulant photosynthetic capabililies signiflCclntly diminished when Ieaves were incubated at 40'C in the clart.~, the mutant PSU toleraled high Lemperalures better thafI the wild type. To obtaîn a 50% decrease in FjFft! it was necessary 10 heat wild-type leaves al 40"C fOf~15 min whereas Ieaves /rom the mutant fl!ached this leveI alter 40 min 01 heat tredtment Similarly, P511-dependent oxygen evoItJ· lions in isolaled thylakoids wefe also higher in mulanl compared to wild-type after brief ex,» sure to high lempmture in the darX (rewll no! shawn). However, extended incubation at 4O"C in the Iight (which is a condition likely to occur during heat Slress) did not reduce 41. below 90% 01 the starting Yalue in leaves cl eitl1er the wild type or the mutant. Notwithstancling these observations, acdima· tien of plants 10 33'( resuJted in differences in the appearance 01 plants and their relative growth measured as the: fresh weight of aerial plants ( Figure <1). the short tenn, prolonge<! incubation of mutant plants al 4°C revealed a very different mutant phenotype 113]. After as liule as 10 days at "·C, newly deYelo~d leaf tissue 01 mulant plants exhibiled chlorosis, which was not eYidenl in wild·type controls. The degree and extent of chlor~is became progressively more pronounced in mutant plants as the Iow.temperature treatment continued. Alter 30 days al40C mest leaves on the mutant plants were pale green and the plants were noticeably smatler than wild-type controls. Measurements 01 chloro· phyU content showed !.hat both mutant and wild-type plants lest chlorophyll at the beginnng 01 the 40C treatmeot w !hat plants of both geootypes contained 309& Iffi chIorophyll after 10 days. Alter this initialloss, the chJorophyll content 01 the wild t~iocrNsed again whiJe the chlorophyll content of the mutant continued to dedine throughout I~cold treatmenL Measurernents 01 FJF'" and lf:l. on leaYeS sampied !rom 4·C-gtM'f'I plants and assayed at 25"C showed that the chlorophylll~s ms accOl'Tlpanied by a decline in photosynthetic !ff1Cieocy (Figult 1). In partkular, lf:l. leU sleadily throughout the experiment to reach 53% oIlhe starting ""lue after 30 days. By conlrasl, FjF'" silowro relatively liltle change during the !irst 5 to 10 days at 4°C and h.1d dedined by less than "" by JO daY'.
These changes in phot~yntheüc performance and chlorophyll content were accompanied by extensive changes in chloroplast ultrastructure in the mutanL Before transfer to low temperature, the thylakoid struclure and organisation of mutant chloroplasls were substantially similar 10 wild type. Wild-type chloroplasu retained this structure even after 30 days at 4°C. HO\vever, the same treatment resulted in extensiye loss of thylakoids .00 50 koid membranes are responsible lor the deatl of triple mutant plants grown at high tempe rature. On mis line, 6oo1eni<add il a pre<:urso of jasmonic: add lhal is involved in many plan responses (29,30].
The nature of the effects seems also 10   1 the absence of trienoic latty acids slabilizes PSU against heat treatmenl applied in lhe oort. In order 10 delerrnioe whether the:se observations are related in acclimated plants, we randomly harvesl.ed leaves dumg gstM'lh al ]]"C, cooled them to 25·C lor 1 hr in the dark and then delermined Fv/Fm (the same result were obtained measuring IIlJ. AI thÎs temperalure, wildtype controls photosyntheti<: efflciency was nol afle<led whereas the quantum yield of PSU eIe<.
tran transport of triple mutant l'las at tirst slighlly inhibitcd from 6 10 9 days but decreased the· reafter to reach l4 and 11 % of the initial value after respectîvely 11